Processes for reproduction of cellular bodies

ABSTRACT

Disclosed are processes for reproduction of cellular bodies of herbivorous and omnivorous mammals. The techniques include obtaining ova, preparation of recipients and transplant techniques. Methods of sex selection of spermatozoa are shown as well as several techniques for clonal production of like embryos.

United States Patent [191 Augspurger PROCESSES FOR REPRODUCTION OF CELLULAR BODIES [76] Inventor: Lynn Lawrence Augspurge'r, 642

Related U.S. Application Data [63] Continuation-in-part of Ser. No. 418,604, Nov. 23, 1973, Pat. No. 3,854,470, and a continuation-in-part of Ser, No. 444,022, Feb. 20, 1974, Pat. 9 No.

[52] U.S. Cl. 128/1; 424/85; 195/18 [51] Int. C1. A61K 17/00; A61K 27/00 [58] Field of Search 128/1 R; 424/85, 100; 195/18 [56] References Cited UNITED STATES PATENTS 3,574,829 4/1971 Wagner et al. 424/100 3,687,806 8/1972 Bovenkamp 128/] R X 3,692,897 9/1972 Bhattacharya et a1 424/85 FOREIGN PATENTS OR APPLICATIONS 7,109,675 1/1973 Netherlands 128/1 R OTHER PUBLICATIONS Symposium on Sex Ratio at Birth, Prospects for Con- [4 Sept. 23, 1975 trol, American Society of Animal Science, 1971.

The Vetinary Annual l969pages 20l2l5Ovum Transplant in Domestic Animals-L. E. A. Rowson.

Reproduction in Mammals-Artificial Control of Reproduction, 1972 Cambridge-Austin & Short-pages 17-31 and 93-101.

Production of Twins in Cattle by Egg Transfer, L. E. A. Rowson, et al.,Journal of Reproduction and Fertility (1971) 25, pages 261-268.

Journal of Reproductive Fertility (1969) 18, pages 517-523, L. E. A. Rowson et al.-Fertility Following Egg Transfer in Cow.

Journal of Reproductive Fertility-Development of Eggs in Rabbit Oviduct-R. A. S. Lawson et al.l972 (28) pages 313-315.

Primary Examiner-Aldrich F. Medbery [57] ABSTRACT Disclosed are processes for reproduction of cellular bodies of herbivorous and omnivorous mammals. The techniques include obtaining ova, preparation of recipients and transplant techniques. Methods of sex selection of spermatozoa are shown as well as several techniques for clonal production of like embryos.

26 Claims, No Drawings PROCESSES FOR REPRODUCTION OF CELLULAR BODIES CROSS REFERENCE TO RELATED APPLICATIONS g This application is a continuation in part of my application Ser. No. 418,604 filed Nov. 23, 1973 now U.S. Pat. No. 3,854,470 entitled Improvements in Reproduction Processes for Cellular Bodies, to which this ap plication is also a Division. This application also is a continuation in part of Ser. No. 444,022 filed Feb. 20, 1974 now U.S. Pat. No. 3,866,598.

In the late 1890s Walter Heape in Cambridge, England, carried out experiments relating to egg transfer in animals. This research has been continued by many experimenters. v

L. E. A. Rowson and his associates at the ARC. Unit of Reproductive Physiology and Biochemistry, Animal Research Station, Cambridge, England, have recently developed techniques for ova transplantation in mammels, particularly in farm animals. The Veterinary Annual, 1969, Bristol, John Wright & Sons Ltd;, has an article by L. E. A. Rowson, pp. 200, entitled Reproduction and Reproductive Disorders", which sets forth their developments. Techniques relating to these developments are also described in Book of the Reproduction in Mammals Series, Artificial Control of Reproduction by C. R. Austin and R. V. Short, published in Cambridge at the University Press, 1972, see chapters one and four; and, one may also refer to the X Biennial Symposium on Animal Reproduction, American Society of Animal Science, as well as Sex Ratio At Birth- Prospects for Control, a Symposium published by the American Society of AnimalScience, 1971. More generally developments in this area are reported in Science (U.S.), Nature (U.K.), Journal of Animal Science, Journal of Reproduction and Fertility and in the journal, The Biology of Reproduction.

These techniques, as developed and described in the above references, employ exogenous gonadotrophins to induce super-ovulation in a donor, sometimes superceded by oestrus synchronization of the donor and the hopeful foster recipients, followed by artificial insemination of the donor, surgical collection of oocytes after fertilization, embryo culture for ashort period of time in commercially available tissue culture medium, and finally transplantation to the uterus of a synchronized recipient of one or more embryos for maturation of the fetus. Rabbits have been shown to be suitable temporary receptacles for the embryo prior to recipient indication, which may be confirmed by a marking transplantation, which is presently preferred to be done surgically, and these receptacles have been used to transport sheep embryos from England to South Africa,

resulting in subsequent-birth from their foster mother. According to Austin and Short, pp. 30, slices ofa rats' ovary, treated with glycerol have been frozen to 79C., thawed and transplanted to recipients. Mice embryos have reportedly also survived freezing to l96C., thawed, and transplanted to recipients, who gave birth to normal and full term living fetusesor new born mice, see Science (U.S.), Vol. 178, pp.411, Oct. 27, 1972. These developments relate to the techniques, as well as other seperately known techniques which may be usefully employed to achieve the resultsdescribed herein. For instance, it is well known thatvasectomy may be performed to make teaser. males as a method of detecting oestrus, as mounting is a reliable methodasdescribed in U.S. Pat. No. 3,076,431 of Feb. 5, 1963.. i

The techniques described herein are believed to represent an improved technique for increasing the reproductive potential of genetically superior cattle, as well as, other form animals and mammals, when the methods, devices and compositions described are employed as indicated.

By employing the techniques described, it may be believed that egg transplantation will become a common practice, and that, contrary to the statement of C. Polge in the aforementioned Artificial Control of Reproduction, it will not be long before the described techniques will be used in a similar way to artificial insemination in order to exploit the genetic potential of the female, the male as well. These techniques can also exploit the true uterine capacity of females, as sheep, cattle and, indeed women, appear to be rather exceptional in that the frequency of large litters resulting from treatment with exogenous gonadotrophins must indicate quite a phenomeal uterine capacity. (C. Polge, pp. 25).

While the foregoing techniques have been used in experimental techniques, they utilize techniques which are not readily reproducible in large numbers and suffer from other difficiencies which are overcome by the improvements disclosed herein.

. While various modifications, combinations and rearrangement will occur to those skilled in the art, both now, and in the future without departing from the scope of the claims appended hereto, it will be appreciated that the improvements disclosed herein make use of available materials or of materials described herein.

Thus, included in the object of the present disclosure are processes to make proctable increased availability of desired genetic material.

Accordingly, disclosed herein are improved techniques in tissue culture of embryos, freezing techniques for oocytes, methods of predetermining the sex of the embroyo which is transplanted use and techniques for clonal bodies so that several embryos of like genetic material may be obtained from the same donor.

The synchronization of donor and recipient is made less critical than hithertofore, permitting greater time delay in transplantation time.

The many improvements which are described hereinafter will be described in particular hereinafter, and consequently the foregoing isnot intended to limit the techniques to be described, not to be an all inclusive description of the improvements which follow.

Among the objects of these improvements are improved processes for the clonal reproduction of a particular animal. The use of sterilized spermatozoa and a pseudo-fertilization solution are used to induce a reaction in an oocyte and the oocyte is caused to become diploid by use of the polar body originally contaminated, transferred or by nucleus transfer.

Addition alclonal embryos are obtained by cell separation. 3

The spermatozoa may be presexed by the various techniques disclosed, and an improvement of this technique is an important part of this application.

Accordingly, a description of the preferred embodiments. follows.

Oestr'us can be determined by observation of mountingjwhich preferably, and most reliably, is done by a vasectomized or gomerized male animals. Such teaser bulls will mount cows at the start of oestrus. Vasectomized males with a portion of the vas defer'ens'removed, or with the removal of the caudidymis, can copulate. By penis and prepuce deviationyeopulation and the consequent transmission of disease-may be prevented. Economics require that teaser bulls be rotated through lots of twenty to thirty cows monthly so as to obtain maxiumum usage while retaining reliability.

Mounting can be observed in close confinement or on the range by trained cowboys. However, a more reliable proof is obtained if the cow is marked by the mounting bull. Kamar, lne. sells the device described in their aforementioned US. Pat. No. 3,076,431. Or the bull may be fitted with a marking halter designed for this purpose and supplied by American Breeders Serviee, DeForest, Wise.

;A preferable means for marking is a topical application of pigmented grease or oil to that portion of the male which contacts the externally'visable rump area of the female during mounting attempts. This may be applied to the brisket and underbelly of bulls.

The marks made upon the mounted female may be readilyobserve d, permitting detection of oestrus even by the untrained. It is desireable that the formulation for the grease be harmless in'topical applications and resistent to being washed off in the rain, but which can be removed with soapy water or detergents. A desireable vehicle is an oil in which a pigmentis mixed. Different colors are desireable to employ, both with respect to providing a daily variation to the color markings.

Examples of desireable vehicles follow:

Formulation Parts/weight Oleic acid or Olive Oil or Peanut Oil 5 0% Lecithin (soybean) l7 Caster Oil 2471 Pigment, as follows: 25%

BluczCobaltous chloride Black: Carbon Black Yellow: Calcium Chromate White: Precipitated Calcium Carbonate Redish: Vitriol red (Red Hematite) Red: Norman Red pigment by the fact that it requires a skilled surgieal technician is the surgical preparation of the male using penis and prepuce deviation, as described in Veterinary Medicine/Small Animal Clinician, April 1973, page 395. Utilizing this method, copulation is prevented.

However, since one of the impostant aspects of this description is to provide a readily reproduceable technique, I prefer the technique of resection of the epididymis, especially in the bovine species. The operation is performed with the bull, shortly before puberty. The operation to be described has a consequent benefit in that the same techniques can be utilized in potential market animals. Bulls grow much more rapidly than steers, and a weight with less fat content 8% greater than that which can be obtained with steers is to be expected.

' the tail of the epididymis is held in position by the duetus deferens and the body of the epididymis, both of which pass dorsally up the side of the testicle. Suture material is used to jointly ligate the body of the epididymis close to the testicle. The tail of the epididymis is severed with scissors and removed. The procedure is repeated for the testicle on the other side. Antibiotic powder is applied to the wound which is closed with sucures or clamps. Wait 20 days before using the bull.

The limited numbers of oceurances of oestrus in a sample group can be increased by synchronization. This can be accomplished by synchronization. The suppression of oestrus by gonadotrophens, particularly progestrogens, is useful in this regard.

Medroxyprogesterone acetate, -C-methyl-l7 acetoxy progesterone, (MAP, Upjohn) is a synthetic progesterone which has been used in an ethanolic solution to suppress oestrus. Melengestrol Acetate (MGA) is marketed by the Tuco Division of The Upjohn Company, Kalamazoo, Mich. 49001, as a food additive available to holders of approved Medicated Food Applications, and is a synthetic progestrogen that is used to suppress oestrus in order to improve rate of gain and feed conversion for feedlot heifers. It is not approved for synv chronization purposes. Presumeably, this is because MGA produces abortions (Journal of Animal Science,

Vol. 30, pp. 433) and the fertilization rate of ova is low 1 while ovarian abnormalities is high (Abstract 155, Journal of Animal Science, Vol. 35.). MGA has been implanted in a polyurethane implant placed in a prominent ear vein or subcutaneously in the ear of heifers. Release rates of 0.4 mg per day have been obtained.

MGA may be fed orally, daily, or incorporated in a grain diet (up to 4.0 miligrams in 1.82 kg of grain per day). 1 prefer to utilize 0.4 mg to 1.0 mg in 1.8 kg daily. The optimal amount may approximate 0.5 mg oral dosage daily. Oral dosages of 0.5mg daily have been shown to suppress oestrus and permit oestrus 2-9 days after withdrawal (Veterinary Medicine/Small Animal Clinician., Vol. 65, May 1970, pp. 491).

Using the preferred oral dosage in grain after withdrawal oestrus will occur 2-11 days after withdrawal with-an average of 4.5 days (i0.5 days) after withdrawal. Heifers and mature females after birth should have a nornal oestrous cycle before medication.

In order to improve the rate of ova fertilization and expected oestrus, on the day of withdrawal 10-20, preferably 15-17 mg, of the steroid Estradiol Benzoate (Arapahoe Chemicals, Inc., Boulder, C010.) is administered in an intra-museular injection. This may be followed by'a similar amount on the 5th day. The first amount may be reduced with a dosage of 15-17 mg on the 5th dayproving satisfactory in most instances. The steroid may be introduced orally by mixture in ethanolic solution and then introduced in saline capsule form, or added to the grain ration.

While fertilization may occur during the first oestrus using the aforementioned technique, in cattle it will be preferred to wait until the second period where conception rates may be improved.

Preferably animals are inseminated on day 26 or 27 and again on day 28 if precise control of detection of oestrus and ovulation described herein is not employed. After synchronization oestrus can readily be detected using teaser bulls. As mentioned above this causes the females to be marked at the start of oestrus.

As previously mentioned, many mammals have a uterine capacity which permits maturation of a greater number of fetuses than is normal in the species. This makes multiple births or twinning especially desireable. However, it is recognized that twinning be desireable, but too many embryos are undesireable. In cattle the preferred method of inducement of twinning is a dose level of 5-15 mg. FSH, preferably 7.5 mg to mg in 3.6 ml of 1% Na carboxymethyl celluose (Abbott Laboratories, Chicago, Illinois) given commencing on day 14-16 of the estrous cycle intramuscularly twice daily for five days. Suppression of oestrus during this period may be desireable and accomplished as previously discusses with the synthetic progestrogens available from Upjohn as previously discussed (MAG). FSH-P available in 10 cc vials of about 50 mg FSH units can be obtained from Armour-Baldwin Laboratories, Omaha, Neb. One-fifth of a vial provides a suitable dosage. FSH tends to overcome the tendency of PMS to produce the undesireable superovulation which may produce too many oocytes for maturation.

It is well recognized that twinning in cattle has disappointing side effects when different sexes are carried, as even one male of a litter will usually produce all female freemartins in the litter. Twinning has the result, however, of increasing the production of animals and the unhappy side affects can be overcome by using the sex control techniques later discussed.

Superovulation may be distinguished from the above twinning techniques, in that usually a greater number of ova are produced at ovulation.

Reproduction of selected individuals may be accomplished to utilize more fully the genetics of desireable females. The techniques of superovulation and transplantation of ova have been generally described. Certain improvements in these techniques and preferred steps will be fully described herein.

Suppression of oestrus is desireable in recipient mammals. The prior description may be referred to again. The cow or heifer that is to be superovulated should have a cycle sychronized with those of recipients, but because of fertility problems the ova should not be recovered until the second oestrus.

Multiple or super-ovulation may be induced by injection of 1500 to 3000 IU (international units) of PMS, pregnant mare serum. 2000 [U is preferred for cattle, while one halfthe amount used for large animals is suitable for swine and sheep. PMS (Ayerest Laboratories) serum may be injected on the 16th day of the oestrus cycle, or between day -18. A single PMS injection induces hyperfollicular development, even in calves 414 weeks of age. It should be followed by an intravenous injection of5 to 10 mg of NIH-LH leutinizing hor mone 5 days after the PMS injection. LH may be obtained from Diamond Laboratories, DeMoines, Iowa (vetrophin), each vial should have 5 mg of NIH-FSH- S4 and NIH-LH-Slactivity. Twelve to twenty-four hours later the ova produced should be exposed to semen if insemination is to be accomplished in the donor animal.

' It has been mentioned that instead of PMS serum, pituitary gonadrophins (FSH) may be substituted to cause twinning. Since these are rapidly dissapated, between the l2th and 18th day of the oestrous cycle, injections of 6-14mg (the lower level will produce fewer follicles which develop) twice daily for approximately five day, are required, given either in the previously de scribed preferred manner or intramuscularly in physiological saline. It has been suggested that Nilevar (norethandrolone-no longer manufactured by Searle) is useful to prevent oestrus during this period (Journal Animal Science, Vol.34, 1972, pp. 77), but it is not desireable and a low level of MGA (.3.5mg) can be used to accomplish this purpose and seems to increase the number of follicles. FSH-P (Armour-Baldwin) is a suitable pitutary gonadrophin, as previously mentioned. 10 mg of prepared porcine FSH may be substituted. LH, eg. NIH-LH-Sl can be used to cause release -at the end of the period.

After the onset of oestrus in cattle, they should be checked every four hours until the end of oestrus is detected and the ovaries should be rectally palpated at four hour intervals until ovulation is detected. Animals should be inseminated, if done internally of the donor, with semen during the latter portion of oestrus.

The follicular development herein contemplated can be increased, especially when utilizing gonadotrophins by feeding a high energy content ration during the period of follicular development, 7-14 days after last oestrus.

This ration should double the weight of the normal grain ration, with the additional weight of the ration being provided by glucose, and other high energy sources as fat (lard) and mollasas.

At the start of this period, estradiol banzoate, in the dosage elsewhere described herein, is also desirable.

It is most desireable to obtain eggs from a superovulated female between 8 and 90 hours after ovulation. If the eggs have been internally inseminated, then the best period is between 50 and 80;hours, and preferably between and hours, at which time the eggs are mainly in the lower reaches of the oviduct before the ampullary-isthmic junction (a contraction before the uterus) and in the upper part of the uterine horn. This leads to a concentration of the oocytes in this local area.

This period is generally acceptable for noninseminated subjects, but eggs will be found higher in the oviduct, even near the fimbriae. Here the best period is between 40 and 80 hours when most of the eggs have reached a point near the ampullary-isthmic junction.

In other animals, for example swine, the transport is even more rapid than the aforementioned conditions occurring in cattle.

within the period flank laparotomy may be performed. The subjects should be taken off feed 24 hours before the surgery and each given an intramuscular injection of about 0.5 ml of propiopromazine hydrochloride (e.g. Travet, Abbott Labs SCI/mg. ml) 25 minutes before anesthesia. Anesthesia can be a procaine hydrochloride injection on both the dorsal and ventral sides of the first, third, and fifth lumbar vertebra, the amount being to ml depending on individual size.

The incision area is shaved and washed with aseptic solution. An incision can be made between the last rib and the external angle of the illium to cm in length. This can be done using a No. 22 Bard-Parker blade in a No.4 Bard-Parker handle. Using an ecraseur a bilateral ovariectomy and salpingectorny is preformed.

Where an oviduct is disected the cervical end of the uterine horn is clamped and seared with a hot iron and the seared site punctured with a blunt needle of a 20-50 ml syringe containing sterile bovine blood serum (Difco, Detroit, Mich.) or prefereably, when the oviduct is removed, TCM199 or, more preferable, the medium described and given as an example herein for embryo culture. The horn is flushed with serum (which may require 20 to 40 ml serum) and the passings collected in a sterile cup watch glass, the flushings passing through the oviduct. The oviduct is accordingly inserted with a tube having a collecting surge chamber for collecting the released oocytes. Aspiration of the tubule will help this process.

Alternate anesthesia includes an initial injection of pentobarbitone sodium followed in 15 minutes with closed circuit Fluothane and oxygen. The incision can be made immediately anterior to the pelvis in the midline through which the uterus and ovaries are withdrawn clamping off the horn. A mid-oviduct incision is made through which the tubulation is inserted. A syringe of 40 ml. is introduced ahead of the clamp and the oviduct flushed in the foregoing manner. The uterus is then returned to the abdomen and the wound sutured using No. 3 catgut followed by interrupted sutures of the skin covered with tape.

Fertilized eggs may be transferred to a foster recipient mother whose oestrus is synchronized with a variation of i- 2 days.

Transplants use the same techniques as before, except that the ovary containing the corpus luteum is identified by rectal palpatation and the eggs are transferred to the adjacent uterine horn by using a Pasteur Pipette connected to a 1 ml syringe. If the corpus luteum is present on each ovary, eggs may be transplanted to both horns. However, unless this is present, twinning is not as likely. Provided that the eggs (blastomers) are sexed it is desireable to transplant two eggs to increase the likelihood of birth, but this may be done without too great fear of freemartins, since one birth for two oocytes is most likely.

Eggs may be kept if fertilized for a short period of time (10 hours) in a dialysis chamber, or incubation unit as later described for a longer period, or they may be transfered-into the utero-tubal junction of a rabbits oviduct which is litigated so that the eggs do not pass into the uterus. This facilitates their recovery when the oviduct is flushed to recover the eggs. Under these conditions, the eggs will divide normally up to about seven days, as is also possible in an incubator.

Care should be taken to be sure the recipient is synchronized to i 2 days, preferably exactly. It usually takes four days after fertilization before the eggs enter the uterus where they can be collected from the cervix entry procedures two be outlined. Also, we should re- Using a pipette, and techniques like those used for artificial insemination, the eggs can be inserted via the cervix in cattle and horses (not swine), but aseptic conditions should be used to prevent purulent cndometritis. Distension of the uterus with carbon dioxide is desireable for non-surgical transfers.

Non-surgical introduction of eggs into recipients, 2 or 3 of 6 cell to early morula stage, is accomplished starting with drawing the eggs along with 0.5 ml medium or serum into an ordinary insemination pipette which is curved at one end, all under aseptic conditions. The pipette is introduced'through a sterile speculum into the cervical canal of the recipient and passed forward along the uterine horn which has had the corpus luteum detected previously by rectal palpation. CO is introduced via the same pipette until the uterus isfully distended. The equipment comprises two flasks, one containing dry ice pieces which are covered with ethanol and which flask is connected to the second flask beneath a water line (the second flask is half filled with distilled water). The upper space of the second flask is connected to a tee fitting, one side of which leads to the pipette and the other side to a rubber balloon which distends as pressure within the uterus increases. Immediately after gassing the pipette is withdrawn and the uterus should remain distended for four to six hours.

Transplantation by these. non-surgical techniques contemplates the use of a flexible cannula made preferably of Silastic (TM of Dow Chemical) tubing in a pipette. The pipette is inserted into the uterine horn and the cannula is passed (through the pipette) to expel the transplant into the uterine horn, or with small size transplants, into the oviduct through the isthmic junction.

Before transfer of fertilized ova to the recipient it will be desireable to sex the embryo being transferred. This can be ,done in a variety of ways. Clearly, the fertilization of the oocyte with ordinary spermatazoawill result in a count of onesex. Yet, since it is possible to maintain the embroyo in vitro for a period of time, as will be described, it is also possible to microscopically determine with an electron microscope whether the X or Y chromozone is present in cells of the embryo.

Cells of the morula stage or the blastocyst are excised from the embryo. With the blastocyst, which is the more desireable in view of the lowest damage risk, the

" zona pellucida is held with a pipette under suction and member that about the same time is used when the eggs are recovered surgically from the oviduct and uterus. Recipients should receive the eggs before regression of the corpus luteum, and preferably within the period ending on the 6th or 7th day.

the trophoblast penetrated with a needle large enough to excise one or two cells.

In order to examine cells for chromozone content, 0.075 KCl (Gibco No. Rl5-0575) is mixed to a total volume of 4 ml and refrigerated. The cells are suspended in 0.5 ml of fetal calf serum and 0.5 ml of hypotonic solution added and followed with 3.5 ml just before centrifugation. Centrifuge for six minutes at 750 rpm. The elapsed time from addition to termination should not exceed 12 minutes. Discard all but 0.25 ml of supernatant and re-suspend the cells in the remaining supernatant. Fix with three parts absolute alchol to one part of glacial acetic acid by first adding 0.5 ml and follow with 3.5 ml of fixative solution. The cells should stand in fixative solution 15 minutes. Discard all but 0.25 ml of fixative supernatant and repeat the fixation procedure.-

After the fixation procedure has been repeated, remove all but 0.5 ml fixative solution and re-suspend the Slaughtered animals may bea source of oocytes which may be transferred to living recipients for maturation. Slaughtered animals are treated prior to slaughter as if normal surgical or non-surgical procedures'are to be adopted. The time of slaughter should correspond to that outlined for surgical techniques. The animal may be slaughtered after fertilization or before insemination as in surgical recovery techniques.

Slaughter is accomplished by stunning the animal and bleeding. While bleeding is being accomplished, an incision is made ventrally and the ovaries, oviducts, and the uterine horns are recovered intact. The uterine horns are immediately clamped and scared. The organs are placed in a transfer incubator to maintain the organs at 30 to 38C., preferably 3 l33C., and transferred to the point where the eggs are to be recovered.

The recovery is accomplished by severing the oviduct and the eggs are flushed from the oviduct by insertion of a syringe through the uterine horn and injecting 2040 ml of serum or preferably tissue culture medium (TCM199 or Ham's F10 or F12, Gibco).

The eggs are deposited in a watch glass and covered with parafine oil. They are examined at 50X and collected and transferred to the preferred tissue culture medium described herein and subsequently to the recipient.

Unfertilized oocytes can also be recovered from the ovary by aspiration of the corpus luteum of the slaughtered animal. These may be fertilized as elsewhere described.

Between recovery and transfer to a recipient it is desireable to culture the embryo in tissue culture medium such as TCM199 or Hams'FlO and preferably the formulations described by way of examples herein. The culture is maintained in a commercial incubator for tissue cultures at temperatures ranging from 31C. to 37C. with 5% CO and 95% air or oxygen. Lower temperatures in the range (3l33C) are preferred. Some success will be had at the normal tissue culture range 37C.

Culture chambers can be glass specimen tubes (30 by 100 mm or 25 by 75 mm) with silicone rubber stoppers.

cone to prevent attachment of the growth to the glass wall. Half of the tube is filled with culture medium buffered with HEPES buffer, and bovine'serum, and

The inner surface of the tube should be coated with silithe other half with the gas mixture which is renewed 7 every eight hours. Tubes should be laid horizontally on rollers and continuously rotated at 30 to 40 revolutions per minute during incubation.

When the eggs are recovered, the flushings are'collected into the tubes. About 3 m1 of fluid is removed at the bottom of each tube after 20 minutes at incubation temperatures and transferred to a watch glass or well slide and the ova is sought under a disecting microscope at 50 X or 187.5X and examined 1875X.

Eggs can then be transferred to the cecipientsin the manner described, or to longer duration culture in tubes or rabbits.

The preferred culture medium, as shown in the following example includes Vitamin B12, Lipoicacid, So-

at ,450X' or dium pyruvate and L-Glutamine. It is buffered with HEPES buffer and NaHCO to a pH of 7.3-7.4.

PREFERRED EXAMPLE CULTURE mgJLiter MEDIUM NaCl. 6800.

Kcl 400. MgSO 7H O 200.

' Na HPO 2H O 60. KH PO 60. Glucose 1800. Phenol red 10. CaCl (anhyd.) 200. NaHCO; (depending on pH) 400.

L-Arginine HCl 70.0 L-Histidine HCI 20.0 L-Lysine monohydrochloride 70.0 DL-Tryptophan 20.0 DL-Phenylalanine 50.0 BL-Methionine 30.0 DL-Serine 50.0 DL-Threonine 60.0 DL-Leucinc 120.0 DL-lsoleucine 40.0 DL-Valine 50.0 DL-Glutamic acid monohydrate 150.0

. DL-Aspartic acid 60.0 DL-Alpha-Alanine 50.0

' L-Proline 40.0 L-Hydroxyproline 10.0 Glycine 50.0 L-Glutamine 100.0 Sodium acetate 50.0 L-Cystine 20.0 L-Tyrosine 40.0 L-Cystcine HCI 0.1 Adenine Sulfate 10.0 Guanine HCI 0.3 Xanthine 0.3 Hypoxanthine 0.3 Uracil 0.3 Thymine 0.3 Disodium alpha tocopherol phosphate 0.01 Thiamine HCI 0.01 Pyrodoxine HCl 0.025 Riboflavin 0.010 Pyridoxal HCl 0.025 Niacin 0.025 Ca pantothenate 0.010 i-lnositol 0.050 Ascorbic acid 0.050 Folic acid 0.010 Para-Aminobenzoic acid 0.050 Feric nitrate Fe(NO,,) 0.100 d-Biotin 0.010 Menadione 0.010 Glutathione 0.050 Vitamin A 0.100 Calciferol 0.200 Tween (Trademark Atlas Powder) 20.0 Adenylic acid 0.200 Adenosinetriphosphate |.0 Desoxyribose 0.5 Ribose 0.5 Choline CI 0.5 Vitamin B 1.3 Lipoic acid 0.2 Sodium pyruvate 1 10.0 L-Glutamine 200.0

Plus 20; fetal calf serum, heat inactivated. per liter of above example.

EXAMPLE 2 Same as above but Earles salts (Gibco) are substituted for the salts of the first example while Glucose is still 1800 mg/L.

EXAMPLE 3 Sameas example two with twice the vitamins and amino acids by weight.

EXAMPLE 4 Same as first example with twice the vitamins and amino acids by weight and with Glucose at 1000.0

mg/L.

Oocytes may be removed from the corpus luteum and matured in vitro. A number needle is attached to a 100 ml syringe filled with 20 ml of culture medium. The corpus luteum is identified by laparoscopy performed 32-36 hours after gonadotrophin injection to cause follicular development or in the first 24 hours of heat or oestrus in cattle, preferably before 14 hours have elapsed after detection.

A 15-20 cm incision is made as described for surgical recovery of ova or mid-ventrally above the udder in cattle. The abdominal cavity is distended with CO and a labroscope inserted to locate the ovaries and the corpus luteum. The corpus luteum is penetrated with the needle and the oocyte aspirated under steady vacuum.

The oocyte is transferred to a watch glass having 5 ml of medium covered with parafin or mineral oil. Cumulus cells surrounding the oocyte are removed with exposure to hyaluronidase for three to five minutes. The oocyte is removed with a pipette and 0.1 ml of medium and washed twice with 2 ml of medium. Care should be used to be sure that the oocytes are always covered with medium or PBS medium (Dulbeccos phosphate buffered salt solution) to prevent dehydration.

Oocytes aspirated from the folicule will mature in vitro when the follicular development has been initiated by gonadotrophin injections as used in causing superovulation and the oocytes are removedfrom the walls of thinned (developed) follicules.

Oocytes will mature when recovered from the corpus luteum of mature follicules in about four hours, as the cumulus cells are seperated. There usually is no need to separate the cumulus oophorus.

Oocytes may be obtained by slaughter 6 to 14 hours after oestrus detection. The oviducts are seperated into Vasections and the unopened corpus luteumshould be aspirated as well. The sections and the aspirated corpus luteum oocytes should be placed in medium covered with parafin oil or mineral oil.

Whenever the oocytes are recovered they should be kept warm in an incubator or on a warm stage of a disecting microscope. The parafin oil should be equilibrated with 5% CO in air in the culture medium.

if necessary the cumulus cell clot should be withdrawn.

Care should be observed to retain an amount of fresh follicular fluid as 0.1 ml of follicular fluid is introduced with 0.4 ml of sperm having a concentration of one to two million per ml. Within two to four hours the cumulus cells surrounding the oocytes seperate and form a layer on the surface. By five to seven hours in concentration the fertilization is complete and the eggs can be placed in culture for maturation to the morula stage or transferred to a recipient.

The fertilization in vitro-utilizes the examples of formulationwof the culture medium buffered to pH7.3 -7.5 with Sodium Bicarbonate and dilute HCl. Sodium pyruvate or sodium oxaloacetate should be part of this fertilization medium and bovine albumen at by volume utilized. The medium for fertilization'and culture should contain 50 milligrams of streptomycin sulfate/ml and 75 mg penicillin G(potassium salt)/ml should also be added. These however should be used when sperm seperation by sex is used.

Synchronization of the donor and recipient as to ovluation time can be overcome by employing improved techniques.

A delay can be achieved by culture of the trans planted cellular bodies for a period of time.

This can be accomplished by the freezing techniques described herein, or by moving back the development of the donor cell by separation of the cell so that the total number of differentiated cells which are transplanted are at the stage that they would be had normal progress been permitted.

Thus, it is preferred to transplant cellular bodies to recipientsat the time corresponding to a few days from the time of division of the first cell or the cellular bodies and the time of normal fertilization of the recipient. The object is to provide the donor cell to the recipient at a time approximately that which permits implantation at the blastocyst stage of development.

This is preferably 12 days of synchronization of the expected blastocyst or morula stage of the embroyo and the date the receipient would expect its own blastocyst or morula stage.

Such timing permits greater use of non-surgical transplantation, as well as surgical transplants at a later time, and should be done before the 10th day after receipient ovulation, preferably before the 7th day after ovulation.

The cell separation techniques and freezing techniques used to accomplish this result are disclosed elsewhere herein.

Sex determination is important for many reasons and can be accomplished by sexing an embryo after fertilization or in other ways as described herein.

Spermatazoa may be presexed by separation of the X and Y chromozone bearing Spermatazoa, and the resultant separation utilized to fertilize matured oocytes.

One method of accomplishing separation is by centrifugation of closed pipettes containing semen at 750 rpm several times. The female sperm will weigh slightly more and they tend to be concentrated at the lower portion of the tube. Spermatozoa should be suspended in a thin buffered solution during centrifugation'. Electrophorisis has been used to separate spermatozoa. Neither of these techniques are particularly satisfactory in the current state of the art.

However, an improved technique using ion exchange materials may be employed as described herein. It is recognized that spermatozoa have a net negative charge, but there appears to be a localization of the charge on either the head or tail of the spermatozoom. Better results appear possible to yield the desired female spermatozoa using ion exchange materials, alone or in combination with centrifugation.

Cations are used to produce entrapment of male sperm, and anions used to entrap female" spermatozoa. This can be done by inducing floculation of the undesired sperm in a watch glass having high surface area to volume, the preferred method being described be- While it is recognized that greensands and zeolites can be used as the ion exchange materials, the more satisfactory are finely divided ion exchange resins. Cation resins of the carboxylic divinyl benzene copolymers or the products of copolymerization of methacylic acid with divinyl benzene and those of maleic anhydride with styrene and divinyl benzene prove suitable. Streptomycin is not used in the support medium with carboxylic resins due to its ion exchange with them. These cation resins will yield spermatazoa capable of producing female offspring.

Anionic exchangers can be produced by nitration and subsequent reduction of styrene divinyl benzene copolymers. These will yield spermatazoa capable of producing males.

The resultant resins have the following structures:

Base Anion Exchange Resin semen in the centrifuge, alone, or in combination with the appropriate ion exchanger, and the dillutent sollution centrifuged at 750 rpm, at a speed lower than 1000 r.p.m.. Here is a second, or third, force applied, namely, the application of a pressure differential of 1530 cm from than at which the sperm was obtained.

For male sellection the above techniques are applied, and the speed is raised to be greater than 1000 rpm.

The resins which are used can be based on Dowex Resins (Trademark Dow Chemical Company, Midland, Michigan). The more strongly basic anion resins of the styrene-divinylbenzene type, Dowex 1,2,21K, can be used, but the polystyrene polyamine Dowex 3 which is weakly bacic can be better used in medium with a pH range up to 7.1-7.3. Mesh size 100 or greater (100-300) is preferred. Suitable resins are B342-AG3, B343-AG3 (both polystyrene polyamines) and Bl43-AG1, Bl44-AGI (polystyrene guaternary amonium) as anions and B544-AG50, B545-AG50 (based on Dowex 50) as cations may be used and obtained from Bio-Rad Laboratories, Richmond, Calif.

Anion and Cation type resins may be of the cellulose type, with the cation type being preferable. A 100 to 200 mesh exchanger of Type CM-cellulose (carboxymethyl cellulose) from Brown Company, 555 Fifth Ave., New York, N.Y., can be used.

To obtain the sample, the semen is washed twice and supported in a buffer solution with pH 7.3-7.5. The resin is introduced into a test tube with a funnel lower end and an enlarged lip, which test tube is in turn supported within a collecting test tube. The sperm is introduced above the resin and caused to pcrcolate through the resin, preferably by centrifugation at 750 rpm for 1 minute or longer. The base of the outer collecting tube has additional buffer support solution to collect the seperated sperm.

The sperm is then Stratified, and the desired spermatazoa removed and used for insemination or frozen.

Another technique contemplated to be used alone or in combination with the aforementioned ion exchange materials for separation of sperm fractions is a combination of centrifugation and pressure differentials.

This technique is especially effective with cattle due to the great difference in size of the X and Y sper matazoa. A conical tube separation chamber ofa coun ter-streaming centrifuge is used. The desired female fraction is obtained by placing washed and dilluted and less than 1200 r.p.m.. A vacuum in the order of l5-30 cm is applied as a differential compared to that of sperm recovery pressure.

The fractions are thus obtained and withdrawn from the dillutent and concentrated. They may then be used for insemination or forzen in a buffer sollution for later use.

The preferred embodiment utilizes counterstreaming centrifugation in combination with an ion exchange material. While the pressure differential appears to improve results, satisfactory fractions may be obtained as otherwise described. Such fractions are used to inseminate recoveredova.

When sex has been determined the blastocyst can be transferred to a recipient and allowed to mature.

Cells of the embryo can have several different operations performed which aid in the reproduction of genetically superior animals and make possible like sexes to be carried by the recipients. This make possible utilizing more fully the uterine capacity of cattle, by avoiding freemartins, and is applicable to other species.

Fertilized ova and small embryos, preferably no greater in size than the morula stage may be frozen to 196C. This is accomplished by washing the embryos in 2 ml of medium, as described herein by example, or with PBS solution. Ova recovered from the corpus luteum should have extraneous cumulus cells of the graphian follicule removed by exposing them to hyaluro'nidase (150 US. pharmacopeia units per milliterlin the medium for 3 to 5 minutes. i

In a very small drop (approximately .01 ml) medium, the eggs are transferred to pipettes containing 0.1 ml of medium. The cooling is done in a bath at a rate less than 2C. per minute with the optimum between 03 and 04C. per minute. At 0C., 0.1 ml of 2 M dimethyl sulfoxide is added as a buffer. After or more minutes the samples are transferred to a bath of approximately 4C. and seeded two minutes later with a minute ice crystal. Samples are then cooled at the same rate to 70C. and then to l 10C (ethanol and dry ice bath) and then transferred directly to liquid nitrogen (-l96C.). Liquid helium can be used to cause the temperature gradient and rate of cooling.

Thawing is done by placing the pipette in a tube 40 X 200 mm in 1 10C. ethonol and warmed by contact with room temperature air at 4 per minute, measured starting 65C. At 0C. then 0.2, then another 0.2 and finally 0.4 ml of culture medium is added. The embryos are then transferred to 1 ml of medium and rinsed twice with a like amount. They are examined at 50X to determine the recovery rate and transferred to culture, e.g

to a watch glass under mineral oil at 30-38C.- (Preferably 31-33C.) in a 5% CO and oxygen or air atmosphere.

The cellular bodies which are thus frozen should be frozen in the preferred medium herein containing sodium pyruvate or sodium oxaloacetate in equal amounts. PBS solution containing these substances may also be used, and the addition of glucose is also desirable. a. i

It is important to increase the utilization of recovered ova and to freeze in the same container embryos of the same sex. I

To this end it is desirable to separate individual cells of one ovum witha lysin, as described herein, and to, place cells of like character in the same container.

The lysin separated cells are fully capable of differentiation after thawing and improved results are obtained in the freezing process.

The zona pellicuda of the morula stageor lower embroyonic cellular body, including those which are separated by lysin treatment, may be desirably removed beforefreezing. i I

After thawing the cellular bodies may be protected by transfer to the zona pellicuda of a second cell.

These procedures are described in more detail elsewhere.

Oospheres maybe frozen in the foregoing manner and thawed to 0C. and successive drops of sperm, preferably with follicular fluid, as later described, mixed with the support medium until the amount of fluid support medium is approximately 0.8 ml to 1 ml and the medium is allowed to warm by exposure to air temperature for 3 hours. For a more detailed description of in vitro fertilization reference may be had to the example given without reference to freezing elsewhere.

The operations on cells, morulas and blastocysts are performed with the aid of a phase contrast disecting microscope. The cell support employed is a small cup shaped watch glass which has been covered and has access aperatures of small side in the sides through which the operating tools may be inserted. These'holes and' the coverplate are sealed with silicone oil film, and the tools may be held with micropositioners and inserted throughthe oil film. The microscope stage should be heated.

Microneedles may be made from l mm Pyrex (Trademark of Coming Class) or borosilica glass rod drawn out to form a thin shaft (about 0.3 mm) 50 mm long/A vertical bend is made to provide clearance between the microtool holder and the stage of the microscope. The thinner shaft is hooked to carry a weight, and then the hooked shaft is loaded with a 1 g. load and drawn out at an angle of 45. The tip is formed with a bead which is then wiped off, or for a hooked needle the tip is cooled slightly and then bent at an angle of approximately 120 so as to form a hooked needle with an included angle of about 60.

' Micropipettes are made like needles starting with 1 mm glass capillaries'Between drawing operations the pipette is hooked, and finally the pipette is also drawn to a very thin portion with a 250 mg load at a 45 angle. The orifice is formed at the surface to form a bevelled bead orifice. Commercial micropositioners (Leitz) may be used to hold the-tools with operations being performed within the cells with the tools thus prepared being held with the micropositioners.

At the two cell stage before division the cells should be closely observed to watch for division to the four cell stage. At the four cell stage, the upper and lower quadrants of the same hemisphere of the four cell stage can be micro-surgically separated by mechanically passiriga glass microneedle through the zona pellicuda (if it has not been previously removed withlysins) and then with slow steady pressure separating the two hemispheres. This procedure may be used to produce identi- Cal twins. i v

Cloning of a fertilized ova is possible. The technique to be utilized herein is to'remove the zona pellicuda at the morula stage'of development or at the 8 to 16 cell stage by trypsination. The cells are suspended in the broth and exposed to the trypsin solution and after 15 minutes mechanically agitated with a glass microneedle to separate individual cells. The cells are washed quickly with phosphate buffered medium.

Concurrently therewith it is desireable to have additional'donee ova available with the zona pellicuda intact. With a sterile needle under the microscope the individual donee cells have their necleus removed or destroyed. The donor cells are aspirated into a needle attached to a'syringe and the cell is injected into the nuclear region of the donee cells, and subsequently, these donee cells are transferred to culture or to the cecipient for maturation. Normally the cells are observed through the first division after the injection before transfer. I p i The zona pellucida can be removed by trypsin and the cells disassociated by trypsination or other lysin.

"Cells maybe suspended in the preferredmedium or in Eagles basicmedium, both modified" to have twice the usual amount of amino acids and vitamins parts/volume), typto se phosphate broth (10 parts/- volume) and bovine'albumen serum l0 parts/volume) in a 1 12 ml flat petri dish having 20 ml medium under parafin or mineral oil. Then 0.5% solution of sodium edetate in a 1% solutionof trypsin is added to the medium. The cells should be incubated for 15-30 minutes until the zona pellicuda is removed.

Dissociation of the cells after the zona pellicuda is removed maybe accomplished by a 0.1% trypsin crystallized once (Worthington Biochemical Corp., Freehold,

17 NJ.) in PBS solution (phosphate bufferedsaline with Eagles salts) and after -30 minutes gently pipetted.v

You can use 0.01 ml of PBS with 0.005 of trypsin solution. Detachment can be aided by stirring or other mechanical help, as by insertion of a fine needle into the cell mass and agitating.

After detachment with trypsin or other lysin, a sterile syringe is filled with about 0.1 ml of medium and the cells removed and washed twice with clean solution to remove the trypsin. One or more of the cells can be sexed with the help of an electron microscope.

If it is desired to chance repeating the above type of cloning, the cells must be permitted to grow again to the morula stage in culture, the cells being placed in 2-4 ml culture medium in a 32 mm plastic tissuecul ture petri dish, and placed in a larger dish containing 1.0 ml of PBS solution and tightly sealed in plastic bags to grow to a larger size. v

However, it should be understood that each repeat process greatly increases the likelihood that incomplete embryos will develop.

It is possible to have development of the separated cells without the zona pellicuda, but the zona pellicuda acts as a natural protection within the reecipient and survival to maturation is thus more easily obtained. As previously stated, this is accomplished by transfer of the donor cell to a donee cell. This is accomplished by aspirating the cell and injecting it into a donee cell which has its zona pellicuda intact and its nucleus destroyed. This is accomplished in various manners, these being described below.

An oocyte is held with a pipette under a disecting microscope. The zona pellicuda is pennetrated with a hooked needle and the necleus is pulled out of the cell or destroyed with agitation. Then into this regionthe donor cell is implanted. The necleus can also be by causing extrusion of the chromozones from the cell if a cell is capapable of division. During metaphase a small drop of silicone oil is placed adjacent the cell, and the cell will extrude the chromozones into the oil where they can be removed. However the mechanical removal of the nuclear protoplasm is preferred, and the use of dividing cells in interphase or the oocyte can be used. It is also possible to withdraw the necleus with a capillary needle by aspiration. By using fine needles the cell deformation caused by thicker needles is avoided.

Another technique for transfer of cellular bodies fron one embryo is to remove the cell from the zona pellucida by lysin application or surgically. Thereafter the removed cell or cells are transplanted to a new zona pellicuda and allowed to develop therein. The necleus of the original cell can be destroyed by ennucleation, destruction of the nucleus, as is preferred, or by excision.

These techniques or combinations thereof can be employed to delay the time of necessary transplant and to achieve more clonal bodies.

The transplated cells can be transplated in theirentirety, or only the nucleus and surrounding cytoplasm can be transplanted to the new cell body. If the latter is used, it is necessary to enneculeate the recipient cell and to transfer the necleus and surrounding cytoplasm into the cytoplasm of the foster cell.

While the above described technique, is simple and expedient. it is also contemplated that alternative techniques can be employed. The donorrcell zona pellicuda can remain intact and cells removed from the blastomereor morula and transferred in the foregoing manner to a donee cell. The cells may be extracted as complete cells and good results may be obtained. However, it is also possible to extract the necleus itself and transfer this necleus to the donee cell. The timing of this last technique is more critical, as both the necleus and the donee cell has a tendency to disintegrate, and the transfer must be accomplished by conducting both operations in parallel or in a short. time.

These techniques produce identical twins" which are not of the same genetic material as the father or mother, containing genes from both, as the genetic characteristics are determined by fertilization before seperation of the embryonic cells.

Female offspring having the same genetic material as the mother animal may be especially desirable.

New individuals have already been created in the lower animals without fertilization by spermatazoa. Turkey eggs have been fertilized with viruses, to'produce male turkeys.

According to two techniques described hereinbelow utlization of the chromozone content of the female alone is contemplated.

Two oocytes are employed. Each oocyte is a secondary oocyte which has elinated a single polar body and reached the mitotic metaphase of the second maturation division, as it is at the time of ovulation.

Both the polar body and the pronucleus are haploid. The two oocytes are placed in a closed disecting chamber on the heated stage of a phase contrast disecting microscope. Pressure within the chamber is carefully monitored. The support medium should be fortified with glucose and in the proportion by weight described in Example I. The support medium may be the same used for trypsinization, with an equal amount of hyaluronidaze and trypzin added. The done e cell is punctured and the vitelline membrane closelyobserved for the formation'of cortical granules. Within 5-10 minutes the pronucleus of the trypsinized second 00- cyte has its necessary development and it is removed and injected into the first donee oocyte. Then equal amounts of Androgamone III, a fatty acid, and sodium dodecyl sulfate and bee venom is added to the support medium. From the equal percentage by weight of trypsin, hyaluronidase and the Androgamone [11, sodium dodecyl sulfate and bee venom, a pseudo fertilization of the donee cell is obtained to some extent.

As a substitute a washed, dense, suspension of spermatazoa may made in the trypsin solution and centrifugedat high speed. The supernatant will contain an extract which is substituted to the same amount by weight as the fatty acids and. detergents at the same point of time in the process.

' The polar body may be substituted for the pronecleus of the second oocyte in a second embodiment.

When metaphase is observed, the result of the joining of the two haploid bodies have fused to form the diploid zygote and the sex of the embroyo is determined by chromozones obtained solely from the mother animal.

When the cell has been observed to have the violent reaction characteristic of fertilization the diploid zygote is removed, washed and transferred to culture medium.

Thereafter the cell is watched to determine whether normal division occurs and thereafter it may be handled as if the cell is a result of normal fertilization.

Living sterile spermatazoa are'capable of initiating the pseudo-fertilization. .Such spermatozoa are obtained by subjecting normal spematozoa to ultraviolet light. The amount of exposure is variable, and depends upon the species, the proximity of the light source and the time of exposure. Too much exposure will kill the spermatozoa. The preferred time of exposure ranges quite widely, as 30 seconds to 3 minutes have yield satisfactory results.

Such sterile ultraviolet irridiated (or sterlized in an- 1 other manner as by exposure to radiation, as is done with fly control programs) can initiate the telophase development.

The expulsion of the polar body would normally result in a haploid body, which while progressing with divisions, will not mature.

In addition to the aforementioned methods of supplying an additional haploid body, alternative approaches may be utilized to produce the desired diploid.

While the normal oocyte development temperature of cattle is between 100 and 102.5 F., the oocytes may be collected and maintained at a lower temperature.

Cattle, swine and horses yield ova which can be cultured at 37 C or as previously mentioned desirable results can be obtained at even lower temperatures in the 30-34 C range and preferably 31 C to 33 C.

Pseudo fertilization can be achieved at these temperatures by the aforementioned process. This should be done under a microscope so that development of the .fertilized ova can be observed. When it is noted that the second polar body is progressing toward but before occurrence of, expulsion through the vitilene membrane, the ova should be subjected to heat shock of approximately C by placing the culture slide in a water bath of the higher temperature. This raises the temperature above normal temperature by below the temperature at which the cell would die.

While yield is quite low, it has been observed that normal diploid bodies do result. These diode in the normal course and can be transplanted for further development. The cells may be cultured and additional clonal copies made by cell separation of the embroyo, as aforesaid.

Yield may be improved by implanting the necleus obtained from another ovum of the same animal, as mentioned before. Heat shock may be used in conjunction with this treatment. This technique may be combined with the use of sterile sperm to produce clonal cellular bodies.

Another example of preselected sperm which may be utilized in fertilization of an oocyte employs the aforementioned technique of separation, preferably through a cation exchange resin, and with a medium containing -40% albumem, preferably bovine albumen (Grand Island Biological Company). Here the aforementioned cation exchange resins should be utilized. The resulting sperm will comprises a greater percentage of female producing sperm.

In practice when attempting to cause clonal copies of the genotype of the donor, the use of presexed but sterile sperm may assure a greater yield of transplantable bodies, since the same unsterile spermatazoa may be effective to cause true fertilization, and the result, while not a genotype of the donor, will be usable as a transplant body.

Reference may be had here to the work of Dr. B. C. Bhattacharya and G. .I. van den Bovenkamp as illustrated by US. Pat. Nos. 3,687,806 of Aug. 29, 1972 and 3,692,897 granted Sept. 19, 1972, which patents are incorporated herein by reference.

In the former patent ordinary sperm of approximately equal X (female causing) and Y (male causing and predominating slightly among most species) chromozones is injected into a rabbit and blood serum obtained in the usual manner and heat inactivated. This serum is then mixed with an intermediate sperm fraction of the desired type (all X) and all of this fraction is agglutinated and the mixture centrifuged to obtain in the supernatent an antibody product which subsequently will be exposed to presumably normal sperm to inactivate all but spermatazoa of the aforesaid desired type, be it X or Y. To accomplish this technique a titer for the specific serum being used must be obtained such that a known amount of serum will agglutinate all spermatazoa in the intermediate fraction and a partial antibody amount of the final inseminate. In this instance it was thought extremely important that all of the desired type spermatazoa agglutinate while leaving the reactive antibody amount which will subsequently inactivate the undesired type. This is stated to require that at least 80-90% of the spermatazoa of the surplus (desired) sperm be agglutinated with the antibodies which are present in a 5OX-50Y ratio in the mixed serum. The resultant product therein claimed will have an excess of antibodies which may be subsequently used to inactivate the undesired type.

The US Pat. No. 3,692,897 differs from the above in that the afforementioned extremely important step of mixing is eliminated entirely. In this case, the sperm fraction is used as the antigen in the rabbit, and the resultant antibodies will agglutinate a like sperm fraction yielding an inseminate used to produce the opposite sex. Using the techniques recommended in this patent a fraction is used as an antigen and the serum produced is directly applied to 50X-50Y ejaculate to inactivate the undesired fraction. Bhattacharya suggests that at least 80 to 90% sperm of an antigen fraction be of the opposite type than that desired to be produced by the ejaculate in order to yield antibodies of the type needed for inactivation of the undesired type.

A summary by E. Schilling, one of the early workers in this field, appearing in the Symposium papers Sex Ratio at Birth- Prospects for Control, published by the American Society of Animal Science, 1971, reviews his own works and that of Dr. Bhattacharya, and concludes that the yield is not to be routinely expected but rather an approxiamate predominance of two-thirds of a particular type is a more likely goal to be achieved.

My prefered fractionalization techniques are those which I disclosed above. In using the immunological enhancement of this seperation, these techniques are also preferred, with the understanding that the enhancements disclosed below are not necessarily limited to the use of the preferred steps of obtaining biased fractions. Alternatives are those disclosed by others, including gravity sedemintation, galvanic seperation, and other techniques disclosed in the Symposium papers, and the references cited therein, provided they do yeild a biased viable fraction.

The preferred immunological enhancement of the seperation techniquesreferred to above'does not utilize, in the preferred embodiment, a substantially pure sperm fraction of a single sex type as an antigen. Rather, contrary to the earlier teachings of others, I prefer to use a normally distributed antigen. Thus the inseminate should be SOX-SOY preferably, and not biased more than firld in either direction. 9 r

The process of obtaining blood serum is of theknown type, as was generally described by Bovenkamp in his patent. The serum is extracted from coagulated blood, withdrawn from a host which has been exposed to the sperm which is utilized as an antigen.

Fresh semen is washed in 1% saline or normal saline to cleanse it by several decantations. Using a hemacytometer having 25 large squares and a 0.1 mm cham{ ber, a count of concentration is taken of 5 squares, which count when multiplied by will yield a concentration count per cc. The decantate should be extended to X 10 (range is normally 10 to 30 X I0 cells per cc. (assuming cattle) with normal saline and 1 cc injected subcutaneously into the host twice a week for the six week period, or otherwise as may be sufficient for the host to develop antibodies of sufficient strenght to be used with a normal inseminate. After the period, the antibody serum is prepared in the normal manner, and the serum is heat inactivated.

Thereafter a clear extender is prepared, as for instance the common egg yolk citrate extender (from which I have for clarity substituted H O for egg yolk by volume). The extension should be approximate 3 times the normal extension (the reciprocal of the smaller fraction used in the inseminate). For example, sodium citrate solution is prepared by dissolving 2.9 gm sodium citrate in 200 ml. ionized distilled water (only 100 ml. if equal egg yolk is used). The amount of extender which is normally required for a single insemination (e.g. 10 X l0 in 1 cc for cattle) is determined and multiplied by the reciprocal of the smallest fraction of sex.

type which is present in the ejaculate to be utilized as Y the inseminate (i.e. 3 ifa Vii-Va fraction is used) and 500 units of penicillin and l mg. streptomycin per ml of the amount utilized added. The amount utilized is then exposed to a normal volume insemination sample by mixing the volumes tygether in an incubation type tube at 37C. To this the serum is added by titration to determine the titer which will agglutinate all of the sample volume.

Thereafter a normally extended equivalent inseminate sample e.g. 10 X 10 normal live cells per cc. of a /a% fraction inseminate, when exposed to the titer volume will be expected to agglutinate or inactivate substantially all of the smaller fraction and an approximately equal amount of the larger fraction. This can be rather simply done by adding the titer to the ampule normally supplied by the breeder services or by drop ping the contents of a straw or standard pellet into a titer vial prepared for this purpose. It is desireable to maintain the titer semen mixture at approximate body temperature (e.g. 37 or within the tissue culture ranges referred to above) for the same period that it took the original testagglutinate to occur. As a conse-' quence all of the antifobides in the serum will be expected to be utilized in immobilization of the undesired .sperm leaving mobile sperm of the desired type'for use in subsequent insemination. The inseminate may be thus used directly, or as a reusable product as suggested" below. In order to raise the concentration of mobile spermatazoa used in the inseminate, it maybe necessary to increase the amount of the titer and sperm by an equal amount. For instance, when a /a% fraction is used 3 times the semen volume and.3 titers volumes are used.

As noted above, the product may also be percipated and reused as an antigen. in thisinstance distilled water-sodium citrate and no egg yolk is used as an extender, or simply saline. When following, after injection of this antigen, the procedures of US. Pat. No. 3,692,897, in spite of the factthat a substantially pure fraction is not utilized as the antigen fraction, equivalent results may be obtained and yield offspring will be of the opposite type as compared with that obtained wehn the product was directly used by mixture with the inseminate.

These seperation techniques are of general applicability. However, the immunological techniques I have disclosed are preferred especially in retarded transplant operations;

When there are not effective antibodies which may affect embryos of a specific sex type, the semen of the same sex as that of the embryo may be employed as the same or relatively close in time period after oestrus, such that the native ova of a cow and a transplant embryo are susceptable to further development as not identical twins. Care must be taken here to not upset the time of implantation of the embryo. If the transplant is at the proper stage of retarded development, then it is possible to inseminate and transplant at like times. This leads consequently to freezing the embryo with the selected inseminate, both being frozen in buffered tissue culture medium as disclosed above, and the utilization of non-surgical techniques disclosed herein.

The result is the susceptibility of these techniques to widespread use, with little additional training required. Proper attention to nutrition levels is however required in order to retain the twin developing embryos, and to prevent retention of the placenta at birth.

I claim:

1. A process for embryo transplantation for increasing the number of progeny of female omnivorous and herbivorous hoofed mammal donors comprising,

inducing superovulation in a desired donor by administration of gonadotrophin, fertilizing ova produced by said donor with a spermatazoa fraction of a desired sex, and

. transplanting selected ova or individual ovum derived from said donor to a selected recipient at the time that the recipient is synchronized with the development of the transplanted fertilized ovum or ova such that the transplant is synchronized at the blastocyst stage in the recipient atthe time that the recipient would normally :implant an ovum of its own had the recipient been fertilized.

2. A differentiated cellular body derived by the process according to claim 1.

3. In a processs for selecting the sex of offspring, the steps of utilizing spermatazoa as an antigen to form antibodies in a host, deriving a serum from said host containing said antibodies, mixing a titerof the serum with quent host in a process for deriving offspring of a desired sex. i

5. The inseminate product of the process according to claim 3.

6. A product according to claim 3 in combination with an embryo suitable for transplantation to a recipient.

7. A process for separation of spermatazoa to produce a fraction containing spermatazoa of a predetermined sex character comprising,

washing sperm and transferring spermatazoa to a dillute fluid support solution,

percollating the spermatazoa through and ion exchange material,

concentrating a fraction of the percollated spermatozoa as the product and transferring the concentrated product to a buffered solution.

8. A process according to claim 7 wherein the ion exchange material is a cation exchange material.

9. A process according to claim 7 wherein the cation exchange material is a resin of the group consisting of carboxylic divinyl benzene copolymers, products of copolymerization of methacylic acid with divinyl benzene, and

products of maleic anhydride with styrene and divinyl benzene.

10. A process according to claim 7 wherein the ion exchangematerial is a cation resin and the dillute fluid is percollated through the resin and the lower fraction utilized to provide the concentrated spermatazoa fraction.

11. A process according to claim 7 wherein the ion exchange material is a resin having the structure 12. A process according to claim 7 wherein the ion ond force is a pressure differential from that pressure at which the spermotazoa was recovered.

17. A method according to claim 15 wherein the centrifugation is at a speed less than 1000 r.p.m.

18. A method according to claim 15 wherein the centrifugation is at a speed greater than 1000 r.p.m. and less than 1200 r.p.m.

19. A method according to claim 16 wherein the pressure differential is l5 to 30 cm.

20. A method according to claim 15 wherein there is an ion exchange material within the centrifuge through which the spermatazoa percollates.

21. A process for embryo transplantation according to claim 1 wherein the spermatazoa fraction is obtained by exposure of spermatozoa to serum containing antibodies to inactivate spermatazoa which will produce an undesired sex. I i

22. A process according to claim 21 wherein the fraction is obtained by the process which includes the steps of utilizing spermatazoa as an antigen to form antibodies in a host, deriving a seru'mfrom said host containing said antibodies, mixing a titer of the serum from said host with a spermatazoa fraction having a predominate fraction of a particular sex type, permitting inactivation of a portion of said predominate fraction, and thus deriving from the residue an inseminate fraction for use in said process.

23. A process according to claim 1 wherein a spermatazoa fraction which is used in the process is ob-. tained by steps including,

washing spermatazoa and transferring said spermatazoa to a dillute fluid support solution, percollating the spermatazoa in said dillute fluid through a collumn containing an ion exchange maexchange material is a resin having the structure terial, and

(|:H3 .1 CH (I: CH H COOH COOH CH CH 13. A process according to claim 7 wherein the percollation is conducted under a vacuum of 15 to 30 cm. differential.

14. A process according to claim 7 wherein the percollation is conducted under a pressure.

concentrating the product fraction of spermatazoa passing through said collumn at a point in time and transferring the concentrated fraction to a buffered solution for subsequent use. 24. The product of the process according to claim 23. 25. A process according to claim 1 wherein a spermatazoa fraction which is used in the process is obtained by steps including,

washing and dilluting the spermatazoa recovered from a donor, placing the dillute solution containing spermatazoa in a counterstreaming centrifuge and centrifuging for a period exceeding twenty minutes, during centrifugation exposing the spermatazoa to a second force, discontinuing centrifugation and removing the desired fraction from the dillutent.

26. The product of the process according to claim 25.

Disclaimer and Dedication 3,906,929.Lynn Lawrence Augspurgez; Birmingham, Mich. PROCESSES FOR REPRODUCTION OF CELLULAR BODIES. Patent dated Sept. 23, 1975. Disclaimer and Dedication filed Oct. 15, 1982, by the inventor."

The term of this patent subsequent to Dec. 17, 1991 has been disclaimed and dedicated to the Public.

[Oflicial Gazette December 28, 1982.] 

1. A process for embryo transplantation for increasing the number of progeny of female omnivorous and herbivorous hoofed mammal donors comprising, inducing superovulation in a desired donor by administration of gonadotrophin, fertilizing ova produced by said donor with a spermatazoa fraction of a desired sex, and transplanting selected ova or individual ovum derived from said donor to a selected recipient at the time that the recipient is synchronized with the development of the transplanted fertilized ovum or ova such that the transplant is synchronized at the blastocyst stage in the recipient at the time that the recipient would normally implant an ovum of its own had the recipient been fertilized.
 2. A differentiated cellular body derived by the process according to claim
 1. 3. In a processs for selecting the sex of offspring, the steps of utilizing spermatazoa as an antigen to form antibodies in a host, deriving a serum from said host containing said antibodies, mixing a titer of the serum with a spermatazoa fraction having a predominate fraction of a particular sex type, permitting inactivation of a portion of said predominate fraction, and to thus derive from the residue of the spermatazoa fraction an inseminate for use in fertilization of ova to yield offspring of which the selected sex is predominate.
 4. A process according to the preceeding claim 3 wherein said residue is reused as an antigen in a subsequent host in a process for deriving offspring of a desired sex.
 5. The inseminate product of the process according to claim
 3. 6. A product according to claim 3 in combination with an embryo suitable for transplantation to a recipient.
 7. A process for separation of spermatazoa to produce a fraction containing spermatazoa of a predetermined sex character comprising, washing sperm and transferring spermatazoa to a dillute fluid support solution, percollating the spermatazoa through and ion exchange material, concentrating a fraction of the percollated spermatozoa as the product and transferring the concentrated product to a buffered solution.
 8. A process according to claim 7 wherein the ion exchange material is a cation exchange material.
 9. A process according to claim 7 wherein the cation exchange material is a resin of the group consisting of carboxylic divinyl benzene copolymers, products of copolymerization of methacylic acid with divinyl benzene, and products of maleic anhydride with styrene and divinyl benzene.
 10. A process according to claim 7 wherein the ion exchange material is a cation resin and the dillute fluid is percollated through the resin and the lower fraction utilized to provide the concentrated spermatazoa fraction.
 11. A process according to claim 7 wherein the ion exchange material is a resin having the structure
 12. A process according to claim 7 wherein the ion exchange material is a resin having the structure
 13. A process according to claim 7 wherein the percollation is conducted under a vacuum of 15 to 30 cm. differential.
 14. A process according to claim 7 wherein the percollation is conducted under a pressure.
 15. A process for seperation of a desired spermatazoa fraction comprising, washing and dilluting the spermatazoa recovered from a donor, placing the dillute solution containing spermatazoa in a counterstreaming centrifuge and centrifuging for a period exceeding twenty minutes, during centrifugation exposing the spermatazoa to a second force, discontinuing centrifugation and removing the desired fraction from the dillutent.
 16. A method according to claim 15 wherein the second force is a pressure differential from that pressure at which the spermotazoa was recovered.
 17. A method according to claim 15 wherein the centrifugation is at a speed less than 1000 r.p.m.
 18. A method according to claim 15 wherein the centrifugation is at a speed greater than 1000 r.p.m. and less than 1200 r.p.m.
 19. A method according to claim 16 wherein the pressure differential is 15 to 30 c.m.
 20. A method according to claim 15 wherein there is an ion exchange material within the centrifuge through which the spermatazoa percollates.
 21. A process for Embryo transplantation according to claim 1 wherein the spermatazoa fraction is obtained by exposure of spermatozoa to serum containing antibodies to inactivate spermatazoa which will produce an undesired sex.
 22. A process according to claim 21 wherein the fraction is obtained by the process which includes the steps of utilizing spermatazoa as an antigen to form antibodies in a host, deriving a serum from said host containing said antibodies, mixing a titer of the serum from said host with a spermatazoa fraction having a predominate fraction of a particular sex type, permitting inactivation of a portion of said predominate fraction, and thus deriving from the residue an inseminate fraction for use in said process.
 23. A process according to claim 1 wherein a spermatazoa fraction which is used in the process is obtained by steps including, washing spermatazoa and transferring said spermatazoa to a dillute fluid support solution, percollating the spermatazoa in said dillute fluid through a collumn containing an ion exchange material, and concentrating the product fraction of spermatazoa passing through said collumn at a point in time and transferring the concentrated fraction to a buffered solution for subsequent use.
 24. The product of the process according to claim
 23. 25. A process according to claim 1 wherein a spermatazoa fraction which is used in the process is obtained by steps including, washing and dilluting the spermatazoa recovered from a donor, placing the dillute solution containing spermatazoa in a counterstreaming centrifuge and centrifuging for a period exceeding twenty minutes, during centrifugation exposing the spermatazoa to a second force, discontinuing centrifugation and removing the desired fraction from the dillutent.
 26. The product of the process according to claim
 25. 